CN104509442A - Method for orientationally cultivating ginseng adventitious roots by dynamic balance alimentation - Google Patents
Method for orientationally cultivating ginseng adventitious roots by dynamic balance alimentation Download PDFInfo
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Abstract
The invention discloses a method for orientationally cultivating ginseng adventitious roots by dynamic balance alimentation. The method comprises the following steps: (1) setting the volume ratio of an primary culture medium to a nutrition additive solution to be (2-3): 1, wherein the primary culture medium is a 3/4SH culture medium+ IBA 3mg/L+ saccharose 20-35g/L, and the nutrition additive solution is a 3/4SH culture medium+ saccharose 90-120g/L; (2) adding the primary culture medium into a bubbling type bioreaction tank, and performing inoculation; (3) feeding air for cultivation; (4) after cultivation is performed after 5-10 days, dropping the nutrition additive solution, and finishing dropping after 3-5 days; (5) continuously cultivating for 30-42 days, and obtaining the adventitious roots. Nutritional components can be dropped in real time according to requirements; the bubbling type bioreaction tank realizes pneumatic stirring under the action of rising bubbles, so that the nutrition additive solution can be fully mixed into a basic culture medium; the adventitious roots can be orientationally obtained, the cultivation time is shortened, and the yield of the adventitious roots and the content of total saponins are increased or the content of target saponins monomers in a component is increased.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method of bioreactor culture ginseng adventitious root.
Background technology
Ginseng (
panax ginsengc. A. Meyer) be Araliaceae (Araliacede) Panax (Panax L.) plant, be one of well-known " northeast Triratna ", the rare Chinese herbal medicine that China is traditional especially, has won fame both at home and abroad.The physician of the Ming Dynasty of China greatness, pharmacy man Li Shizhen (1518-1593 A.D.), in its epic monumental work Compendium of Material Medica one book, have been done detailed introduction to effect of ginseng, have been established the good reputation of ginseng " kings of hundred grass ".Ginseng is grown on that day and night temperature is little more, height above sea level is the softwood forest of 500 ~ 1100 meters or in the hillside of mixed forest, its main producing region is mainly distributed in 40 ° ~ 45 ° (N), between 117.5 ° ~ 134 ° (E), China be mainly distributed in three provinces in the northeast of China and northern Hebei mountain region area, especially with Mountain area, Changbai for main producing region.China is research one of ginseng country the earliest, describes ginseng in detail and have the effect of " tonifying five zang organs, peace spirit, determine soul, only palpitation with fear, except perverse trend, eyesight-improving intelligence-developing, take to make light of one's life by commiting suicide for a long time and prolong life " in the pharmacology monograph Shennong's Herbal of earliest extant
[7].Modern pharmacology research shows, ginseng have reinforce vital energy, multiple arteries and veins takes off admittedly, reinforce the spleen to benefit the lung, promoting production of body fluid and inducing sedation of the mind effect, ginseng has anti-arrhythmia, anti-hemolysis, immunity moderation system, regulates many pharmacological activities such as central nervous system simultaneously
[8].The chemical constituent more complicated contained in ginseng, except containing except ginsenoside, also containing the active component of multiple non-saponin(e in ginseng, as containing carbohydrate (monose, oligosaccharides, oligosaccharide, polysaccharide etc.), amino acid, fatty acid, brass, vitamin (VB1, VB2, VB12, nicotinic acid, pantothenic acid etc.) and wet goods component of volatilizing.Along with modern pharmacology in recent years, the deepening continuously of pharmacokinetic, medical value and the health-care efficacy thereof of ginseng obtain certification further, constantly rise violently in price and the status of ginseng, nowadays ginseng is not only as the rare Chinese herbal medicine of China, obtain the extensive accreditation of the people of the world especially.
Ginsenoside has good pharmacologically active and health-care efficacy, shows that ginsenoside has good pharmacological activity for cardiovascular and cerebrovascular, central nervous system, immune system, digestive system etc. through pharmaceutical research.And very active for the research of monomer ginsenoside pharmacological activity, the rare ginsenoside that particularly some content are very micro-, has high medical value.
Because ginseng is regarded as treasure for a long time, wild resource is exhausted, and artificial cultivation occupying woodland, causes water and soil loss, and the cycle is long, management cost is high, disease is serious.
The advantage that ginseng-cell is cultivated, root is cultivated be its huge biosynthesis ability there is primordial plant root having, root cultivate in the height differentiation of cell the synthesis capability of secondary metabolite is significantly improved, show out obvious metabolic stability simultaneously.
The Fiber differentiation technology of ginseng adventitious root reaches its maturity, generally applies, by fresh of perennial ginseng clean, sterilization, cutting after containing 2,4-D, KT and sucrose MS solid culture medium in carry out callus induction.Again the callus induced is moved to induction in the MS of IBA and sucrose, propagation adventive root.
Cultivate compared with ginseng-cell, the advantage that root is cultivated is its huge biosynthesis ability having primordial plant root and have, in root cultivation, the height differentiation of cell makes the synthesis capability of secondary metabolite significantly improve, and shows out the stability of obvious metabolic stability and vigor simultaneously.
Ginseng adventitious root bioreactor culture, generally apply sucrose carbon source is provided, along with the increase of sucrose concentration, the output of ginseng adventitious root also increases thereupon, and in medium sucrose concentration 60g more than L, have inhibitory action to the growth of adventive root.
At present for bioreactor culture propagation ginseng adventitious root, mostly adopt and will prepare that medium is disposable to add in reactor, inoculate adventive root, be cultured to till nutrition exhausts.Results adventive root.And in the process of bioreactor culture adventive root, be a dynamic process of growth, growing the demand of each stage to nutrition is not quite similar, and the nutrition of medium is a process reduced gradually theoretically.Disposablely add medium, specifically can not adapt to the needs of each vegetative stage.
Have report to adopt same medium, add in reactor at twice or three times, number of times is more, and growth coefficient is less.
Summary of the invention
The object of the invention is, for solving bioreactor culture ginseng adventitious root, the unbalanced problem of each Stage Nutrition, to improve the output of ginseng adventitious root and the content of secondary metabolite, and provide dynamic equalization alimentation to train the method for ginseng adventitious root for a certain area.
The method of dynamic equalization alimentation post-directed training ginseng adventitious root, it comprises:
1) initial medium and nutrition annex solution volume ratio are 2 ~ 3 ﹕ 1; Medium+IBA 3 ~ 5mg/L+ sucrose 20 ~ 35g/L based on initial medium; Medium based on nutrition annex solution+sucrose 90 ~ 120g/L, sterilizing; Described basal medium is 3/4 ~ 4/4 SH or 3/4 ~ 4/4 MS medium;
2) initial medium is added in bubble type biological reactor, inoculation;
3) ventilate cavity;
4) cultivate by 5th ~ 10 days, drip nutrition annex solution;
5) continue to cultivate by 30th ~ 42 days, results adventive root;
Described basal medium is 3/4SH medium;
Described IBA is butyric acid potassium;
The sucrose of described initial medium is 20 ~ 30g/L;
Optium concentration is 30g/L;
Described nutrition annex solution also comprises: methyl α-naphthyl acetate 1.5 ~ 2.7mg/L and/or methyl jasmonate 150 ~ 240mg/L and/or salicylic acid 150 ~ 240mg/L;
Described cultivation temperature is 23 ± 2 DEG C.
Another object of the present invention is to provide the bio-reactor that dynamic equalization alimentation cultivates ginseng adventitious root, and it comprises: flow container 6 is added in initial medium retort 4, air pressure pump 5 and nutrition; Initial medium retort 4 is bubble type biological reactor, is provided with pneumatic means bottom it, and the shower nozzle of pneumatic means is many micropores shower nozzle 405, and reaction cover 403 is also provided with nutrition annex solution and drips mouth 401; Air pressure pump 5 is connected with pneumatic means; Be provided with band control valve discharging opening 64 bottom described nutrition interpolation flow container 6, top is provided with cover 62 and band filtering air inlet 61, air inlet 61 is located on cover 62, and nutrition annex solution is dripped mouth 401 and is connected with discharging opening 64 by pipeline 7;
The diameter that described nutrition annex solution drips mouth 401 is 2.0 ~ 0.2mm;
The air inlet 61 that flow container 6 is added in described nutrition is connected by air inlet regulating device with air pressure pump 5.
Another object of the present invention is that dynamic equalization alimentation cultivates ginseng adventitious root system, it comprises: flow container 6 and 3 initial medium retort 4 are added in 1 nutrition, and the nutrition interpolation discharging opening 64 of flow container 6 drips mouth 401 with the foster annex solution of 3 initial medium retort 4 and is connected.
The air inlet 61 that described initial medium retort 4 inlet duct, nutrition add flow container 6 is connected with air pressure pump by pipeline.
Described bubble type biological reactor 4 and nutrition are added flow container 6 and are 18L, and initial medium addition is 8-10L, and the addition of nutrition annex solution is 12-15L.
The invention provides the method for dynamic equalization alimentation post-directed training ginseng adventitious root, 1) initial medium and nutrition annex solution volume ratio be 2 ~ 3 ﹕ 1; Initial medium is 3/4SH medium+IBA 3mg/L+ sucrose 20-35g/L; Nutrition annex solution is 3/4SH medium+sucrose 90-120g/L; 2) initial medium is added in bubble type biological reactor, inoculation; 3) ventilate cavity; 4) cultivate by 5th ~ 10 days, drip nutrition annex solution, within 3-5 days, drip off; 5) continue to cultivate by 30th ~ 42 days, results adventive root.Nutrition can be dripped in real time as required, bubble type biological reactor is under the bubble risen, achieve pneumatic stirring, nutrition annex solution fully can be mixed in basal medium, orientable acquisition is shortened incubation time, the raising output of adventive root, the content of total saposins or is improved the content of target saponin monomer in component.Particularly can use the medium of high concentration sucrose, the sucrose concentration of dynamic equilibrium retort, the addition that can strengthen sucrose improves adventive root output, solves the problem that sucrose concentration height suppresses adventive root growth.Initial medium retort and nutrition are added flow container and are 18L, and initial medium addition is 8-10L, and the addition of nutrition annex solution is the culture systems of 12-15L, for ginseng adventitious root provides best growing environment.
Accompanying drawing explanation
Fig. 1 is the initial medium retort schematic diagram of the bio-reactor of dynamic equalization alimentation post-directed training ginseng adventitious root;
Fig. 2 is that flow container schematic diagram is added in the nutrition of the bio-reactor of dynamic equalization alimentation post-directed training ginseng adventitious root;
Fig. 3,4 is dynamic equalization alimentation post-directed training ginseng adventitious root system schematic.
Embodiment
the acquisition of embodiment 1 ginseng adventitious root
Choose the 6 years raw garden ginsents in Changbaishan area (An County, fair, Tonghua, Jilin Province), stripping and slicing, with MS medium evoked callus, by induction callus with 34 SH medium+IBA5.0mg/L+ sucrose 30g/L, inducing adventitious root, squamous subculture.
the preparation of embodiment 2 initial medium and nutrition annex solution
1, the preparation of medium
1) initial medium
3/4SH medium+butyric acid potassium (K-IBA) 3mg/L+ sucrose 30g/L
2) nutrition annex solution
Nutrition annex solution (1):
In often liter of 3/4SH medium, add methyl α-naphthyl acetate (C
12h
10o
2) 2.7mg/L, methyl jasmonate (C
13h
20o
3) 240mg/L, salicylic acid 240 mg/L, sucrose (C
12h
22o
11) 90g × 3/L.
Nutrition annex solution (2):
In often liter of 3/4SH medium, add methyl α-naphthyl acetate (C
12h
10o
2) 2.7mg/L, methyl jasmonate (C
13h
20o
3) 204mg/L, salicylic acid 240mg/L, sucrose (C
12h
22o
11) 120g/L.
embodiment 3 bio-reactor dynamic equalization alimentation post-directed training ginseng adventitious root
1) added by initial medium 12L in 18L bubble type biological reactor, nutrition annex solution 6L adds in liquid replenisher, bio-reactor and liquid replenisher sterilizing under temperature 120 DEG C, pressure 0.08Mpa1 condition;
2) adventive root cuts 1cm block, is inoculated in biological reactor;
3) ventilate, 23 ± 2 DEG C of cultivations;
4) cultivate by the 10th day, drip nutrition annex solution (1) and drip off for 6L, 3-5 days;
5) cultivation 42 days is continued, results adventive root.
embodiment 4 bio-reactor dynamic equalization alimentation post-directed training ginseng adventitious root
1) added by initial medium 10L in 18L bubble type biological reactor, nutrition annex solution 5L adds in filling tank, bio-reactor and filling tank sterilizing under temperature 120 DEG C, pressure 0.08Mpa1 condition;
2) adventive root cuts 1cm block, is inoculated in biological reactor;
3) throughput, 23 ± 2 DEG C of cultivations;
4) cultivate by the 5th day, drip nutrition annex solution (2) 2L to biological reactor, drip 1 day, cultivate the 10th day, drip nutrition annex solution (2) 3L, within 4 days, drip off; Throughput is strengthened during dropping.
5) cultivation 42 days is continued, results adventive root.
the bio-reactor of embodiment 5 dynamic equalization alimentation post-directed training ginseng adventitious root
Refer to Fig. 1,2, dynamic equalization alimentation cultivates ginseng adventitious root
'sbio-reactor, it comprises: initial medium retort 4, air pressure pump 5 and nutrition add flow container 6; Initial medium retort 4 is bubble type biological reactor, pneumatic means is provided with bottom it, pneumatic means comprises many micropores shower nozzle 405, retort pneumatic means interface 406,, air cleaner 407, mass air flow sensor 408, air intake control valve 409, reaction cover 403 and band filter exhaust mouth 402 are established in top, band filter exhaust mouth 402 is located on reaction cover 403, and reaction cover 403 is also provided with nutrition annex solution and drips mouth 401; Air pressure pump 5 is connected with pneumatic means;
Be provided with band control valve discharging opening 64 bottom described nutrition interpolation flow container 6, top is provided with cover 62 and band filtering air inlet 61, air inlet 61 is located on cover 62, and nutrition annex solution is dripped mouth 401 and is connected with discharging opening 64 by pipeline 7;
The diameter that described nutrition annex solution drips mouth 401 is 2.0 ~ 0.2mm;
The air inlet 61 that flow container 6 is added in described nutrition is connected by air inlet regulating device with air pressure pump 5.
During use, close each terminal valve, 10L initial medium is added in initial medium retort 4, nutrition is added flow container 6 and is added 5L nourishing additive agent, the discharging opening 64 of nutrition interpolation flow container 6 drips mouth 401 with retort nutrition annex solution and is connected, put into the sterilizing of high-pressure sterilizing pot inner high voltage, be inoculated in bubble type biological reactor 4 in desinfection chamber, move to culturing room, start air pressure pump, open band filter exhaust mouth 402 and air shut-off valve, air after filtration sprays through many micropores shower nozzle 405, adventive root is suspended in medium, air is discharged through exhaust outlet 42, 23 ± 2 DEG C of cultivations, cultivate in 5-10 days, open band filtering air inlet 61 and band control valve discharging opening 64 that flow container 6 is added in nutrition, regulate charge flow rate and discharging opening control valve, control rate of addition, drip nutrition annex solution, drip off within 3-5 day.
embodiment 6 dynamic equalization alimentation post-directed training ginseng adventitious root system
Refer to Fig. 1,2,3,4, dynamic equalization alimentation cultivates ginseng adventitious root system, it comprises: flow container 6 and 3 initial medium retort 4 are added in 1 nutrition, and the nutrition interpolation discharging opening 64 of flow container 6 drips mouth 401 with the foster annex solution of 3 initial medium retort 4 and is connected.
The air inlet 61 that initial medium retort 4 inlet duct, nutrition add flow container 6 is connected with air pressure pump by pipeline;
Described bubble type biological reactor 4 and nutrition are added flow container 6 and are 18L, and initial medium addition is 8-10L, and the addition of nutrition annex solution is 12-15L.
the directed alimentation of embodiment 7 bio-reactor dynamic equalization cultivates the mensuration of ginseng adventitious root output and saponin content
1, with embodiment 4,5 for experimental group, with 3/4SH medium+IBA5mg/L+ sucrose 50g/L for medium usual manner cultivate for contrast, found that:
Control group is by the 10th day from cultivation, and adventive root increasess slowly, but 10-20 days rapid developments, within 20-30 days, also show a rising trend, but slower; Within 30-40 days, almost do not find to increase.
Cultivate ginseng adventitious root method by embodiment 4 bio-reactor dynamic equalization alimentation and cultivate adventive root, by the 10th day from cultivation, within the 10-20 days, increase than control group and significantly accelerate, 20-25 days a little more than control group, 25-40 days with increase hardly.
Cultivate ginseng adventitious root method by embodiment 5 bio-reactor dynamic equalization alimentation and cultivate adventive root, each stage comparatively contrasts and significantly improves with embodiment 4 growth, until 35 days have growth trend, within 35 days-42 days, increases hardly.
Embodiment 4 comparatively control group dry weight slightly improves, and nearly 5%, embodiment 5 improves 20%.
2,6 weeks ginseng adventitious roots will be cultivated by embodiment 4 method, and send Ginseng In Jilin Province inspection center to detect, the results are shown in Table 2
Claims (7)
1. the method for dynamic equalization alimentation post-directed training ginseng adventitious root, it comprises:
1) initial medium and nutrition annex solution volume ratio are 2 ~ 3 ﹕ 1; Medium+IBA 3 ~ 5mg/L+ sucrose 20 ~ 35g/L based on initial medium; Medium based on nutrition annex solution+sucrose 90 ~ 120g/L, sterilizing; Described basal medium is 3/4 ~ 4/4 SH or 3/4 ~ 4/4 MS medium;
2) initial medium is added in bubble type biological reactor, inoculation;
3) ventilate cavity;
4) cultivate by 5th ~ 10 days, drip nutrition annex solution;
5) continue to cultivate by 30th ~ 42 days, results adventive root.
2. the method for dynamic equalization alimentation post-directed training ginseng adventitious root according to claim 1, is characterized in that: described basal medium is 3/4SH medium.
3. the method for dynamic equalization alimentation post-directed training ginseng adventitious root according to claim 1, is characterized in that: described IBA is butyric acid potassium.
4. the method for the dynamic equalization alimentation post-directed training ginseng adventitious root according to claim 1,2 or 3, is characterized in that: the sucrose of described initial medium is 20 ~ 30g/L.
5. the method for dynamic equalization alimentation post-directed training ginseng adventitious root according to claim 4, is characterized in that: the sucrose of described initial medium is 30g/L.
6. the method for dynamic equalization alimentation post-directed training ginseng adventitious root according to claim 4, is characterized in that: described nutrition annex solution also comprises: methyl α-naphthyl acetate 1.5 ~ 2.7mg/L and/or methyl jasmonate 150 ~ 240mg/L and/or salicylic acid 150 ~ 240mg/L.
7. the method for dynamic equalization alimentation post-directed training ginseng adventitious root according to claim 6, is characterized in that: described cultivation temperature is 23 ± 2 DEG C.
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| CN105961159A (en) * | 2016-05-11 | 2016-09-28 | 东北师范大学 | Gen-seng cultivation method applying cultivation matrix and nutrient solution |
| CN106538394A (en) * | 2016-12-20 | 2017-03-29 | 江西宜信堂医疗科技有限公司 | A kind of Ramulus et folium taxi cuspidatae culture medium and preparation method thereof |
| CN112920986A (en) * | 2021-04-20 | 2021-06-08 | 山东安然纳米实业发展有限公司 | Method for producing ginsenoside by fermenting ginseng stem cells |
| CN113025555A (en) * | 2021-04-20 | 2021-06-25 | 山东安然纳米实业发展有限公司 | Method for separating and culturing ginseng stem cells by using biological reaction device |
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| CN115053810A (en) * | 2022-06-29 | 2022-09-16 | 珲春华瑞参业生物工程股份有限公司 | Culture method for simultaneously increasing contents of multiple ginsenosides in ginseng adventitious roots |
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| CN105961159A (en) * | 2016-05-11 | 2016-09-28 | 东北师范大学 | Gen-seng cultivation method applying cultivation matrix and nutrient solution |
| CN105961159B (en) * | 2016-05-11 | 2019-07-02 | 东北师范大学 | A kind of culture of ginseng method using cultivation matrix and nutrient solution |
| CN106538394A (en) * | 2016-12-20 | 2017-03-29 | 江西宜信堂医疗科技有限公司 | A kind of Ramulus et folium taxi cuspidatae culture medium and preparation method thereof |
| CN113046292A (en) * | 2021-04-02 | 2021-06-29 | 山东安然纳米实业发展有限公司 | Method for culturing ginseng stem cells by using biological reaction device |
| CN113046292B (en) * | 2021-04-02 | 2022-06-28 | 山东安然纳米实业发展有限公司 | Method for culturing ginseng stem cells by using biological reaction device |
| CN112920986A (en) * | 2021-04-20 | 2021-06-08 | 山东安然纳米实业发展有限公司 | Method for producing ginsenoside by fermenting ginseng stem cells |
| CN113025555A (en) * | 2021-04-20 | 2021-06-25 | 山东安然纳米实业发展有限公司 | Method for separating and culturing ginseng stem cells by using biological reaction device |
| CN113025555B (en) * | 2021-04-20 | 2023-01-24 | 山东安然纳米实业发展有限公司 | Method for separating and culturing ginseng stem cells by using biological reaction device |
| CN112920986B (en) * | 2021-04-20 | 2023-02-07 | 山东安然纳米实业发展有限公司 | Method for producing ginsenoside by fermenting ginseng stem cells |
| CN115053810A (en) * | 2022-06-29 | 2022-09-16 | 珲春华瑞参业生物工程股份有限公司 | Culture method for simultaneously increasing contents of multiple ginsenosides in ginseng adventitious roots |
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Effective date of registration: 20181228 Address after: 133000 No. 3 District of Hunchun Border Economic Cooperation Zone, Yanbian Korean Autonomous Prefecture, Jilin Province Patentee after: Hunchun Huarui ginseng biotechnology engineering Limited by Share Ltd Address before: 130022 11/F, Block B, Hengye Plaza, 1119 Nanhu Road, Changchun City, Jilin Province Patentee before: Jilin Prov. Inst. of Green Food Engineering |